Technical Abstract:
The genetically engineered plum ‘HoneySweet’ (aka C5) has proven to be highly resistant to Plum pox virus (PPV) for over 10 years in field trials. The original vector used for transformation to develop ‘HoneySweet’ carried a single sense sequence of the full length PPV-CP gene, yet the resistance mechanism of ‘HoneySweet’ was found to be based on post-transcriptional gene silencing (PTGS). Sequencing of the transgene insert revealed an inverted repeat of the PPV-CP sequence in ‘HoneySweet’. We hypothesized that this structure, acting as a hairpin (hp), was responsible for PTGS of the transgene and of viral CP which resulted in a high level of resistance to PPV infection. In order to test this hypothesis, the hpPPV-CP insert was cloned from ‘HoneySweet’ and transferred into ‘Bluebyrd’ plum seedlings through Agrobacterium tumefaciens transformation of hypocotyl slices. Two versions of the hpPPV-CP insert were tested. One spanned the CP inverted repeat only (minimal construct), and the other spanned the inverted repeat plus 304 bp of plum DNA upstream and 591 bp downstream of the inverted repeat. Transgenic plum plants containing single or multiple copies of these hp inserts were inoculated with PPV D isolated from Pennsylvania, USA. PPV infection was evaluated through three cycles of cold-induced dormancy (CID) by symptom expression and by at least two and up to five ELISA and PCR tests. Of 24 plants evaluated, nine were never infected, six in some tests showed weak infection, and nine plants were consistently infected. Most of the resistant lines contained a single copy of the minimal hp construct. This data strongly suggests that the hp portion of the PPV-CP insert in ‘HoneySweet’ plum is responsible for PPV resistance.